Method for determining the existance and/or the monitoring of a pathological condition in a mammal

ABSTRACT

There is disclosed a method of testing a cellular hematologic fluid derived from a mammal to determine the existence in the mammal of a pathological state or condition wherein an immunomodulator is admixed with the cellular hematologic fluid of the mammal and a reaction parameter determined and compared with known reaction parameters of cellular hematologic fluids of mammals of known healthy states to like immunomodulator. In a preferred embodiment of the present invention, the reaction parameter is a clotting parameter as determined as fibrin levels.

RELATED APPLICATIONS

This is a continuation-in-part application of U.S. application Ser. No.734,799, filed May 16, 1985, now U.S. Pat. No. 4,705,756 issued Nov. 10,1987, which is in turn a continuation-in-part of U.S. application Ser.No. 703,120, filed Feb. 19, 1985, now abandoned which is a continuationapplication of U.S. application Ser. No. 06/538,783, filed Oct. 4, 1983,now abandoned, which is continuation-in-part application of U.S.application Ser. No. 06/440,540, filed January 26, 1983, now abandoned.

FIELD OF THE INVENTION

This invention relates to a method of testing a hematologic fluid, andmore particularly to a method of testing to determine the existence inthe mammal of a pathological state or condition, to monitor a knownpathological state existing in a mammal and a test kit therefor.

DESCRIPTION OF THE PRIOR ART

Common diagnostic tests performed on asymptomatic individuals during thecourse of their annual physical examiniation might include: completeblood count (CBC), blood chemistries (e.g. glucose or electrolytelevels) and urinalysis (test for glucose, ketones, etc.). Occasionally,these tests may detect a disease which was not obvious upon physicalexamination alone. These routine screening test would be useless indetecting at an early stage the disease states which kill and disablethe great majority of individuals including cancer, rheumatic diseases,AIDS, heart disease, vascular disease, and others. Such disease statescan in part be characterized by abnormalities in either the bloodcoagulation or immune response system, or both.

At present, the detection in a mammal of a pathological state orcondition, e.g. cancer, AIDS, sepsis and the like is generally performedafter the mammal has experienced some abnormal physical response, e.g.lack of energy, headaches, rectal bleeding, lumps, etc., or aspreliminarily detected during an annual physical examination. Onceevidencing such abnormal physical response, diagnostic procedures and/orother protocols are thereafter initiated and evaluated to qualify thepathological state as well as to quantify the extent of advancement ofthe pathological state or condition. Diagnostic procedures may involveX-rays, e.g. mammography for breast cancer, proctoscopy of the colon,etc.

Additionally, once a pathological state has been found to exist in themammal and has been qualified as to the specific pathological state,there may be remedial procedures to reduce the impact of the pathologicstate on the mammal, e.g. drug, radiation therapy, chemotherapy, and thelike protocol, or alternately to eliminate the pathological state, e.g.by surgical procedure. In any event, the effectiveness of the remedialprocedure is difficult to timely assess. For example, in the surgicalremoval of cancerous growth, only subsequent biopsies of proximatetissue may demonstrate total removal, and then, not necessarily on a 100percent certain basis, let alone the possibility of metastasis.

Tests have been developed to determine the immune function of monocytes,neutrophils, lymphocytes, etc. wherein the individuality is isolated andtested for individual functionality by diverse methods. Such proceduresare costly and time consuming and are not specific to a particularpathological state. Also, the results of individuality tests aredifficult to interpret, let alone correlate. For example, althoughmammography may delineate the size, location, etc. of a lump in thebreast in a female, the results will not always qualify whether the lumpis cancerous or benign. Such pathological evaluation is effected bypathological observation of the actual cellular structure after biopsyor surgical removal of the lump.

Some of the above tests or procedures performed in a clinical laboratoryare useful in the monitoring of certain diseases, e.g. liver enzymes forliver disease, blood urea nitrogen for kidney disease, T-cell functionfor immunological disorders, prothrombin and partial thromboplastintimes for bleeding disorders, etc. However, such tests cannot determineeither the effects of therapy on the coagulation changes in thromboticdiseases, or similar effects of therapy in cancer and other diseaseswhich involve alterations in the immune response system.

OBJECTS OF THE INVENTION

An object of the present invention is to provide a method fordetermining whether a pathologic state or condition exists in a mammal.

A further object of the present invention is to provide a method fordetermining whether a pathological state or condition exists in mammalthat may be performed in a facile and inexpensive manner.

Another object of the present invention is to provide a method fordetermining whether a pathological state or conditions exists in amammal, that may be readily effected in a relatively short period oftime.

Yet another object of the present invention is to provide a dependablemethod for determining whether a pathological state or condition existsin a mammal with minimal, if any, false readings.

A still further object of the present invention is to provide a simplemethod for sequentially determining the course of a known pathologicalstate or condition in a mammal.

Still yet another object of the present invention is to provide a methodfor determining effectiveness of a surgical procedure on a mammal toerradicate a pathological state or condition, or to detect recurrentdisease.

Another object of the present invention is to monitor the effectivenessof a drug regime or like protocol on a mammal having a knownpathological state or condition.

Still another object of the present invention is to monitor theeffectiveness of a remedial program for retarding growth, reducing ordestroying a known pathological state or condition in a mammal.

SUMMARY OF THE INVENTION

These and other objects of the present invention are achieved byadmixing an immunomodulator as defined herein and a cellular hematologicfluid of a mammal and determining a reaction parameter thereof andcomparing such reaction parameter with known reaction parameters ofcellular hematologic fluids of mammals of known healthy states with likeimmunomodulator. In a preferred embodiment of the present invention, thereaction parameter is a clotting parameter as determined as fibrinlevels or a function of a time differential between fibrin levels.

In another embodiment of the present invention, the ratio between thereaction parameters of such a cellular hematologic fluid of a mammalbeing tested without and with an immunomodulator is compared with theratio between the reaction parameters of cellular hematologic fluids ofmammals of known healthy states without and with a like immunomodulatorto assess the existence or non-existence of a pathological state orcondition in the mammal being tested.

DETAILED DESCRIPTION OF THE INVENTION

It has unexpectedly been observed that a reaction parameter of acellular hematologic fluid of a mammal with a pre-existing pathologicalcondition when admixed with an immunomodulator is different than thereaction parameters of cellular hematologic fluids of mammals, in knownhealthy states when admixed with the same immunomodulator. Generally,the method of the present invention does not diagnose a specificpathological condition, but points to the existence of a pathologicalcondition in the mammal being evaluated, although in certain instancesthe process of the present invention may be capable of diagnosingalgorithmically a specific pathological condition. As used herein,cellular hematologic fluid of a mammal is the whole blood thereof or afraction thereof including monocytes and other cellular or noncellularcomponents of the mammal.

While the theory of the invention is not fully understood, nor doApplicants wish to be held to any theory of invention, it is believedthat blood coagulation and/or the immune response system of a mammalhaving an existing pathological state or condition to an immunomodulatoris different than the blood coagulation and/or immune response system ofhealthy mammals to like immunomodulators. While monocytes to varyingdegrees are involved in the immune response system of the hematologicfluid to the immunomodulator, it is believed that the immune responsesystem involves an interreaction between the monocytes and othercomponents, e.g. T-cells, lymphocytes, neutrophils, etc. in the cellularhematologic fluid.

The pathological conditions, the nonspecific existence of which areidentified by the present invention include cancer, sepsis, AIDS,diabetes, multiple sclerosis, acute myocardial infarction, trauma,vascular thrombosis, etc. and any pathological state or conditionaffecting the immune response system of a mammal, it being understood byone skilled in the art that the specific pathological state or conditionexisting in a test mammal is generally qualified after a positivedetermination of the existence of a pathological state or condition inaccordance with the method of the present invention. The term "mammals"as used herein includes homo sapiens, and domesticated animals, e.g.race horses.

As used herein, the term "immunomodulator" means an immunoactivator orimmunoattenuator which is an agent that either promotes or accelerates,or retards or attenuates, respectively, coagulation of whole blood orwhole fractions (i.e. as expressed by recalcification time (RT)).Immunomodulators include, inter alia, endotoxins, measles virus,Interferon, phorbol esters, collagens, anticoagulants such as warfarin,platelet activating factors, carrageenans, adjunct peptides,thromboplastins, antigens, myeline, gram negative bacteria, lectins suchas Concanavalin-A, mitogens such as pokeweed mitogens, etc.

While there exists a plethora of reaction parameters that may beevaluated in the method of the present invention, it has been found thatthe clotting parameter as determined by fibrin formation, hereinafterreferred to a recalcification time (RT), is a particularly facile andinexpensive method for evaluating a response of a cellular hematologicfluid to an immunomodulator. The term "recalcification time (RT)" isdefined as any time period between initiation of fibrin formation to anend point thereof or to some intermediate point, it being understoodthat values for clotting parameters may be based on the rate of fibrinformation, for example, as defined by the integrated area beneath a ratecurve between limits, etc.

Anticoagulants for whole blood or fractions thereof include the citratessuch as sodium citrate, the oxalates, sodium ethylenediaminetetra-acetic acid, etc., with sodium citrate being generally preferred.

As hereinabove discussed, it has been observed that there exists adifference between the reaction parameters of cellular hematologicfluids of healthy mammals to an immunomodulator, compared to reactionparameters of cellular hematologic fluid of a mammal having apre-existing pathological condition to such an immunomodulator. Thus, inthe context of clotting parameters, and specifically recalcificationtimes, a comparison thereof readily identifies a mammal having anexisting pathological condition. Many algorithms may be developed usingsuch reaction parameters, and more specific algorithms may be derived tomore fully evaluate clotting parameters to determine the existence in amammal of a specific pathological condition or state.

A more sophisticated algorithm is based upon the calculation of a"Thrombotic Index", defined as the ratio of the recalcification time(RT_(v)) of the cellular hematologic fluid of a mammal (in a vehicle,e.g. saline) in the absence of an immunomodulator, to therecalcification time (RT_(i)) thereof also in a vehicle and in thepresence of an immunomodulator, in accordance with the followingequation (I):

    TI=RT.sub.v ÷RT.sub.i

with the thrombotic index of the mammal being tested being compared withthe thrombotic indices of healthy mammals.

Still another algorithm is formulated by a percent difference ofclotting (PDOC) in accordance with the following equation (II): ##EQU1##The percent differences of clotting of test mammals are then comparedwith percent differences of clotting of healthy mammals.

There are many apparatuses available for measuring reaction parameters,e.g. chromatographic columns for concentrations of a specific chemical,as well as for measuring clotting parameters. For example, a SONOCLOT™Coagulation Analyzer is available from Sienco, Inc. for measuringviscoelastic properties as a function of mechanical impedance of thesample being tested. Such analysis is very sensitive to fibrin formationthereby providing improved sensitivity and reproducibiltiy of results.There is another device, the Thromboelastograph (TEG) for similarlymeasuring viscoelastic properties, however the TEG is not as sensitiveas the SONOCLOT® and presents disposal and cleaning problems. Stillanother apparatus is the HEMOCHRON® 400 available from the InternationalTechnidyne Corporation of Edison, New Jersey, evidencing significantdata correlation to that of the SONOCLOT®.

To facilitate an understanding of the present invention, the followingdescription thereof will initially be particularized with reference tothe use of an endotoxin, specifically E. coli endotoxin (strain 055:B5)as the immunomodulator in a suitable vehicle, e.g. saline, on therecalcification time-endotoxin (RT_(i)) of cellular hematologicalfluids.

TESTING PROTOCOL

From a mammal to be tested, there is withdrawn a hematological sample,e.g. by venipuncture using a syringe (20 gauge needle) without stasis orundue force to draw blood. It will be appreciated by one skilled in theart that traumatization of blood sampling should be minimal sinceimperfect sampling introduces tissue factors into the blood sample andthus would impact on the validity of the results. The hematologicalfluid is transferred to a tube including and admixed with ananticoagulant, e.g. 3.8% solution of buffered sodium citrate. Generally,the volumetric ratio is from about nine (9) parts hematological fluid toone (1) part anticoagulant. While many anticoagulants are available,sodium citrate is generally preferred since the pH level thereof isessentially similar to the pH level of the hematological fluid of themammal being tested, and is less toxic to the cellular elements.

Thereafter, an aliquot portion (2 milliliters) of the anticoagulatedhematological fluid or citrated while blood (CWB) is admixed in a tubewith the endotoxin (e.g. 20 μl of a 1 mg/cc suspension of solution of E.coli endotoxin) and incubated for a predetermined time period, generallyof from 2 to 4 hours. It has been generally found that longer incubationtime periods provide result of greater sensitivity.

Generally, incubation temperatures range from about 35° C. to 40° C.,preferably about 37° C. After incubation, a predetermined amount of acalcium-ion containing composition, such as calcium chloride (CaCl₂),e.g. 10 μl of 0.5M CaCl₂ is admixed with 0.4 cc of the incubatedhematologic fluid with the admixture introduced into a cuvette forinsertion into the hereinabove mentioned SONOCLOT® Coagulation Analyzer[100 μl of 0.1M CaCl₂ to 0.5 cc for a HEMOCHRON® 400] set to determine arecalcification time between initial fibrin formation and a "given"fibrin concentration, e.g. a 10% scale deflection as taken as an endpoint. If a thromboelastograph is used, the recalcification time is interms of "R" valves. It is understood by one skilled in the art thatcalcium ions are necessary to initiate fibrin formation.

Recalcification Times-Immunomodulator (RT_(i)) --Mammals in HealthyState

Recalcification times-immunomodulator (RT_(i)) of a cellularhematological fluid for mammals in a healthy state range between 3.93 to6.04 with a mean recalcification time being 4.66, as determined by TEG;and 4.6 to 7.2 with a mean of 5.69, as determined by SONOCLOT®.

Recalcification Times-Immunomodulator (RT_(i)) --Mammals With aPathological Condition

Recalcification times-immunomodulator (RT_(i)) of a cellularhematological fluid for a mammal having a pathological condition, assubsequently confirmed by other diagnostic procedures, range above orbelow the RT_(i) values of healthy mammals, as more fully hereinafterdisclosed and discussed.

EVALUATING PROTOCOL

Comparison of the recalcification times-immunomodulator (RT_(i)) of thecellular hematologic fluid of a test mammal with known recalcificationtimes of cellular hematological fluids of healthy mammals permits anessentially instantaneous evaluation of the state of the test mammal,i.e. healthy or the existence of a pathological condition in the testmammal, as more fully hereinafter discussed with reference to theExamples.

Statistical analysis is used to gather and summarize data in order tomake such data comprehensible and to be able to draw appropriateconclusions from the results. Discussion of the following Examplesincludes certain statistical analyses for a better understanding of theinventive contribution hereof. As used herein, the term "statisticallysignificant differences between the groups studied" means that whenusing the appropriate statistical analysis (e.g. Chi-square tests,t-test) the probability of the groups being the same is less than 5%,e.g. p<:0.5. In other words, the probability of obtaining the sameresults on a completely random basis is less than 5 out of 100 attempts.

EXAMPLES OF THE INVENTION

The following Examples are illustrative of methods of the presentinvention, and it is understood that the scope of the invention is notto be limited thereby. Additionally, it will be understood by oneskilled in the art that a particular pathological state was generallydetermined after a tested mammal (homo sapiens) exhibited a positiveresponse to a method of the present invention. Additionally, the datawith respect to healthy mammals as to a select immunomodulator at givenlimits to obtain recalcification times-saline (RT_(v)) andrecalcification times-endotoxin (RT_(i)) established a base or standardfrom which the mammals being tested were generally compared forrecalcification times-endotoxin (RT_(i)), thrombotic index and percentof different of clotting.

EXAMPLE I PATHOLOGICAL CONDITION--CANCER

The normal volunteer controls were of both sexes, ranged in age from 21to 69, and were both smokers and non-smokers. No data was ascertainedfrom the volunteers as to current drug intake nor whether the volunteerswere currently under treatment for any disease.

The following Table I sets forth mean values and ranges for RT_(i),RT_(v), TI and PDOC of a group of cancer patients and a group of healthyvolunteers (control):

                                      TABLE 1*                                    __________________________________________________________________________    GROUP                                                                              RTi,                                                                              range                                                                              RTv,                                                                              range                                                                              TI, range                                                                              PDOC,                                                                              range                                    __________________________________________________________________________    Control                                                                            4.66                                                                              3.93-6.04                                                                          6.13                                                                              5.24-7.61                                                                          1.32                                                                              1.15-1.53                                                                          23.4 14.0-34.5                                (n = 23)                                                                      Cancer                                                                             2.25                                                                              1.54-4.02                                                                          6.46                                                                              4.45-8.72                                                                          2.71                                                                              1.62-4.39                                                                          60.3 38.3-88.0                                (n = 25)                                                                      p < 0.001         p = NS                                                      __________________________________________________________________________     *Recalcification times determined by TEG.                                

The cancer patients were evaluated at the time of diagnosis of thedisease. There were significant differences between the recalcificationtimes-endotoxin (RT_(i)) between the group of the healthy volunteers andthe group of the cancer patients, whereas there were no significantdifferences between recalcification times-saline (RT_(v)) of suchgroups. Additionally, it can be readily seen that the thrombotic index(TI) is greater for the group of cancer patients than the thromboticindex (TI) of the healthy volunteers. The same proposition held true ofthe comparison of percent difference in clotting (PDOC) therebetween.The values for TI or PDOC do not overlap for these groups.

It has been found in cancer patients where a large portion of the tumorload is removed and minute portions remain, that the RT_(i), TI and PDOCdifferences still range outside the parameters of the group of healthyvolunteers. A patient having cancer of the colon exhibited TI and PDOCvalues of 1.91 and 47.6%, respectively, one week post surgery. It wassubsequently determined that tumor growth had invaded adjoining tissue.

The method of the present invention permits a clinician to evaluateeffects of therapy on the state of the cancer in a cancer patient. Forexample, small changes in RT_(i) values and a lowering of TI or PDOCvalues after non-fully curative treatments have been demonstrated. Ifchemotherapy and/or radiation treatment do not alter such values,changes in the treatment are then suggested to find a more effectivedrug regime and/or radiation protocol. An advantage of the method of thepresent invention is the convenience of sampling and evaluation atvarying times after therapy and the assessment of effectiveness oftreatment prior to physical appearance of clinical changes.

Of the types of cancers detected by the method of the present inventionincluded are cancers of the lung, breast, biliary tract, bladder,larynx, ovary, head and neck, colon, rectum, esophagus, soft palate,pancreas, and floor of the mouth. As hereinabove mentioned, the presenceor absence of remaining malignancy after curative surgery may bedetermined by the methods of the present invention.

EXAMPLE II PATHOLOGICAL CONDITION--BREAST CANCER

The following Table II sets forth specific data with reference to sixpatients; patients 1 to 4 having cancer and patients 5 and 6 havingbenign breast lesions.

                  TABLE II*                                                       ______________________________________                                        Patient    RT.sub.i                                                                             RT.sub.v   TI   PDOC                                        ______________________________________                                        1          2.00   7.31       3.66 72.6                                        2          3.28   7.04       2.15 53.4                                        3          2.51   6.61       2.63 60.2                                        4          3.52   6.06       1.72 42.0                                        5          4.32   4.54       1.05 4.84                                        6          4.87   5.98       1.23 18.6                                        ______________________________________                                         *Recalcification times determined by TEG.                                

The above date clearly illustrates a lower recalcificationtimes-endotoxin (RT_(i)) for the patients (#1-#4) with breast cancer asdistinguished from the patients (#5-#6) with benign breast lesions. Asimilarity of comparison was noted between the RT_(i) values of patients(#1-#4) and RT_(i) values set forth in Table I, above. Additionallypatients (#1-#4) had significantly higher TI and PDOC values.

Patients #3 and #4, post one week surgery, exhibited TI and PDOC valuesof 1.25 and 1.39 and 20.0% and 27.9%, respectively, indicative of thesuccessful removal of all malignance as demonstrated by subsequenthistological examination of tissue and non-existence of cancerous cellsin the lymph nodes.

As hereinabove discussed, in cancer cases where the malignancy is nottotally removed, the RT_(i), TI and PDOC values remain in thepre-operative ranges.

EXAMPLE III PATHOLOGICAL CONDITION--DIABETES

The following Table III sets forth mean values for RT_(i) and RT_(v) ofa group of 36 patients having diabetes:

                  TABLE III*                                                      ______________________________________                                        Group    RT.sub.i  Range     RT.sub.v                                                                              Range                                    ______________________________________                                        Control  5.69 ± 0.74                                                                          4.6-7.2   6.55 ± 0.08                                   5.3-8.5                                                                       (n = 19)                                                                      Diabetics                                                                              4.99 ± 1.20                                                                          3.0-8.1   5.65 ± 2.3                                                                         3.3-16.8                                          p < 0.001           p < 0.001                                        ______________________________________                                         *Recalcification times determined by SONOCLOT ® Coagulation Analyzer.

Eighteen of 36 (50%) diabetics had accelerated clotting in the salineincubated samples and 15 of 36 (42%) had clotting times shorter than theshortest control value for the endotoxin incubated samples. It wouldappear that diabetics with abnormal values have the more severe disease(e.g. juvenile diabetics, diabetics with vascular complications,diabetics with disease more than 15 years, etc.). The methods of thepresent invention will be useful in measuring therapeutic effects ondiabetic activity, such as diet control, exercise and drug treatment,and may become a goal of the therapy to bring the RT_(v) and RT_(i)values of diabetics into the normal range.

EXAMPLE IV PATHOLOGICAL CONDITION--ACQUIRED IMMUNE DEFICIENCY

SYNDROME (AIDS)

The following Table IV sets forth mean values and ranges of RT_(i),RT_(v), TI and PDOC of healthy volunteers (per Example I) with meanvalues and ranges of RT_(i), RT_(v), TI and PDOC of four (4) confirmedadvanced AIDS patients:

                                      TABLE IV*                                   __________________________________________________________________________    Group                                                                              RT.sub.i,                                                                         range                                                                              RT.sub.v,                                                                         range                                                                              TI, range                                                                              PDOC,                                                                              range                                    __________________________________________________________________________    AIDS 4.94                                                                              4.43-5.40                                                                          5.78                                                                              5.12-6.94                                                                          1.17                                                                              1.08-1.29                                                                          14.5  7.5-22.2                                Control                                                                            4.66                                                                              3.93-6.04                                                                          6.13                                                                              5.24-7.61                                                                          1.32                                                                              1.15-1.53                                                                          23.4 14.0-34.5                                __________________________________________________________________________     *Recalcification time determined by TEG.                                 

From the above data, it can be seen that an evaluation of therecalcification times-endotoxin (RT_(i)) of the AIDS patients arehigher, but not necessarily significant as distinguished from likecomparison of cancer patients. It is noted, however, that the TI andPDOC values are significantly lower, particularly in three out of fourcases. Another form of algorithm could be derived to more unequivocallyidentify all such AIDS patients. Thus, the methods of the presentinvention illustrate that mammals having AIDS or AIDS-like disease havea PDOC value below the control, and with some statistically determinedbase line value, to qualify individuals for blood donation.

EXAMPLE V PATHOLOGICAL CONDITION--SEPSIS, TRAUMA, AND/OR SURGERYPATIENTS

In surgical intensive care units, particularly patients exposed to severtrauma and/or major surgery and are being managed by life-supportsystems frequently succumb to multi-system failure resulting fromprogressive and perhaps undetected sepsis.

The following Table V sets forth values of group (9 patients) exhibitinga localized septic condition from a group (62 patients) who did notexhibit such a condition, it being noted that RT_(i) values are lowerand TI and PDOC values are higher, as expected (compared to a normalpopulation) as a result of released thromboplastins and immunologicalconsequences from the presence of traumatized tissues:

                  TABLE V*                                                        ______________________________________                                        GROUP    RT.sub.i                                                                             RT.sub.v                                                                              TI        PDOC                                        ______________________________________                                        Control  4.66   6.53    1.32      23.4                                        Septic                  8 < 1.30 > 2                                                                            8 < 23.0 > 2                                Non-Septic              2 < 1.30 > 60                                                                           2 < 23.0 > 60                                                       p < 0.001                                             ______________________________________                                         *Recalcification times determined by TEG.                                

As hereinabove mentioned, as a result of the traumatic condition of thepatients being tested, the RT_(i), and TI values are not readilycomparable with values of RT_(i) and TI of non-surgery of pre-surgerypatients.

In eighty percent (80%) of the septic patients, the thrombotic indicesare not elevated, as expected. Values of TI lower than 1.30 and PDOCvalues lower than 23.0% usually shown the presence of sepsis in theabdominal cavity of the patient, probably due to the fact that highconcentrations of an immunomodulator have entered the bloodstream ofsuch individuals.

EXAMPLE VI PATHOLOGICAL CONDITION--IMMUNOCOMPETENCE

The following Table VI sets forth the Rt_(i), RT_(v), TI and PDOC valuesfor post-operative and post-traumatic patients for non-survivingpatients (9), surviving patients (46), and non-operative normalvolunteers (23):

                  TABLE VI*                                                       ______________________________________                                        Group       RT.sub.i                                                                             RT.sub.v   TI   PDOC                                       ______________________________________                                        Control     4.66   6.53       1.32 23.4                                       Non-Survivor                                                                              4.51   6.72       1.49 32.9                                       Survivor    2.21   6.09       2.75 63.9                                       ______________________________________                                         *Recalcification times determined by TEG.                                

There were no significant differences in the RT_(v) values, but highlysignificant differences in the RT_(i), TI, and PDOC values betweensurvivors and non-survivors. Unlike the elevated value of TI and PDOC incancer patients, elevated values occurring within 48 hourspost-operative or post-trauma patients are good prognosticationindicators. If the post-operative values are low, there is an indicationof possible septic complications. Further, a TI value of 1.60 appears tobe the threshold value of immunocompetence, since 8 of 9 patients whodied had values below 1.60, whereas only one patient with a value above1.60 died.

The test measures the immunocompetence of an individual. Therefore,activation (accelerated clotting) in response to test endotoxin, of thecancer patient (early diagnosed), and post-operative or post-traumapatients, each showing an altered (activated) state of the immuneresponse system as reflected in accelerated clotting time under theinfluence of test endotoxin, may in part explain the thromboticcomplications associated with these states.

EXAMPLE VII PATHOLOGICAL CONDITION--MULTIPLE SCLEROSIS

The following Table VII sets forth values of 33 patients with multiplesclerosis, many of whom were in clinical remission at the time oftesting:

                  TABLE VII*                                                      ______________________________________                                        Group  RT.sub.i  Range   RT.sub.v                                                                              Range PDOC                                   ______________________________________                                        MS     3.67 ± 1.27                                                                          1.2-7.0 4.99 ± .93                                                                         2.7-7.3                                                                             26.2 ± 20.0                         Patients                                                                      (n = 33)                                                                      Control                                                                              5.69 ± .75                                                                           4.6-7.2 6.55 ± .82                                                                         5.3-8.5                                                                             12.2 ±11.1                          (n = 19)                                                                      ______________________________________                                         *Recalcification times determined by SONOCLOT ® Coagulation Analyzer 

Twenty-two of the 33 MS patients exhibited abnormal RT_(v) values, and29 of the 33 patients had abnormal RT_(i) values. The data indicatesthat an active disease state is present, even though a state of clinicalremission is observed. It is readily appreciated by one skilled in theart that a practitioner will utilize the methods of the presentinvention to tailor medical therapy to the presence of an activedisease, as well as to monitor the patient's condition during therapy.

As indicated earlier and as illustrated above, the test method of thisinvention is capable of detecting a variety of disease states in whichthe latent procoagulant generation is elevated (activated monocytes).These states include myocardial infarction, stroke, infections, acquiredimmune dysfunction syndrome (AIDS), rheumatoid arthritis, cancer,multiple sclerosis, etc.

In addition, by varying the concentrations of the immunomodulators,e.g., endotoxin, carrageenan, etc., the sensitivity to a thresholdchallenge can be ascertained. In some states cancer, multiple sclerosis,etc., use of immunomodulator concentrations one-hundredth the optimumconcentration can also detect an activated cell in a blood sample. Useof these varying immunomodulator does concentrations enables thedetection, and effects of therapy on some diseases.

There are a variety of immunomodulators than can activate isolatedmonocytes and thereby stimulate these cells to generate a procoagoulantactivity in culture. These stimulants include endotoxin, immunecomplexes, complement split products, inflammatory particles, amines,phorbol esters, lectins, antigens, chemical mediators of inflammation,lipoproteins, virus damaged cells, tumor cells, etc. It is believed thatthe stimuli that activate monocytes interact with cellular plasmamembranes. Perturbation of these membranes can occur viaimmunomodulatory-cell membrane contact involving specific receptors orunspecific interactions.

EXAMPLE VIII COMPARATIVE TESTS WITH ADDITIONAL IMMUNOMODULATORS

The purpose of these examples is to show that a multitude ofimmunomodulators in this protocol in which whole blood is employed canbe utilized and that endotoxin was just a representative of the group.

The citrated blood samples utilized to obtain the following data wereform normal individuals as well as from individuals with a variety ofdisease states. In some examples, differences can be seen in the valuesobtained between saline, endotoxin and other immunomodulators but theymay not be significant (p values) changes. This is due to the relativelysmall sample size and/or the fact that bloods from healthy and varyingdisease states were utilized.

In Table VIII, below, the number of citrated whole blood samples tested,the mean RT saline ±SD (standard deviation of the means) RT endotoxin±SD and RT immunomodulator ±SD are tabulated. The analysis of variance(ANOVA) was used when comparing the means for the different group valuesof P-0.05 are significant. PG,22

                                      TABLE VIII                                  __________________________________________________________________________    Number of                                                                     Samples                                                                             Concentration                                                                         Saline   Endotoxin                                                                              Immunomodulator                                                                             Catagory                        __________________________________________________________________________    3     10 μg/cc                                                                           6.43 ± 1.70                                                                         5.00 ± .85                                                                          5.23 ± .68 Adjunct peptide, muamyl                                                       peptide                                                         Adjunct Peptide                                                                             immunological activator, 10                                                   μg/cc                                                                      CWB                             9     20 μg/cc                                                                           6.97 ± 1.94                                                                         5.29 ± .93*                                                                         5.36 ± 1.51                                                                              Lectin                                                          Con A                                         9     20 μg/cc                                                                           4.90 ± 1.28                                                                         4.57 ± 1.07                                                                         4.57 ± 1.32                                                                              Mitogen                                                         Pokeweed                                      9     unknown 4.90 ± 1.28                                                                         4.57 ± 1.07                                                                         4.79 ± 1.03                                                                              Measles virus                                                   Virus                                         7     2 μg/cc                                                                            5.30 ± .91                                                                          4.09 ± .57*                                                                         5.30 ± 1.04                                                                              Platelet activating factor,                                                   lipid,                                                          PAF           mediator released during                                                      trauma                                                                        and inflammation, allergy       5     20 μg/cc                                                                           6.04 ± 1.26                                                                         5.22 ± .49                                                                           4.54 ± .45**                                                                            Zymosan - immunomodulator                                       Zymosan                                       7     1 μg/cc                                                                            5.51 ± .74                                                                          5.94 ± .63                                                                          5.10 ± .87 Myelin, protein, antigen                                        Myelin                                        __________________________________________________________________________    Number of                                                                           Final   RT       RT       RT                                            Samples                                                                             Concentration                                                                         Saline ± SD                                                                         Endotoxin ± SD                                                                      Immunomodulator ± SD                                                                     Catagory                        __________________________________________________________________________    5     1 μg/cc                                                                             .56 ± 1.33                                                                         5.10 ± 1.92                                                                         5.86 ± 1.34                                                                              12-O--tetradecanoyl                                                           phorbol                                                         TPA           13-acetate (TPA phorbal                                                       ester) cellular activator       3     .001 unit/cc                                                                          4.37 ± .76                                                                          3.50 ± 2.35                                                                         3.56 ± 0.72                                                                              Thrombin - enzyme                                               Thrombin      procoagulant                    7     2.0 units/cc                                                                          4.91 ± .51                                                                           4.13 ± 1.08*                                                                        3.72 ± 1.27**                                                                           Interferon -                                                    Interferon    immunologicalmediator                                                         pharmacological agent           9     10 μg/cc                                                                           4.80 ± 1.14                                                                          4.22 ± 0.84*                                                                        3.28 ± 0.68**                                                                           Carrageenan - inflammatory                                      Carrageenan   mediator, colloid               9     400 μg/cc                                                                          4.80 ± 1.14                                                                          4.22 ± 0.84*                                                                        3.55 ± .72**                                                                            Latex Beads,                                                    Latex Beads   phagocytic particle             8     10 μg/cc                                                                           5.26 ± 1.58                                                                          3.04 ±  1.20*                                                                       4.35 ± 1.70**                                                                           Platelet activator,                                             Collagen      abnormal surface for                                                          blood contact,                                                                immonomodulator                 5     3.0 mg/cc                                                                             5.18 ± .88                                                                          4.26 ± .78*                                                                         6.62 ± 1.62                                                                              Amine, intracellular                                            ammonium      compartment modifier                                            chloride      affects pH.                     __________________________________________________________________________     *signifies RT endotoxin different than RT saline.                             **signifies RT immunomodulator different than RT saline.                 

In addition to the above immunomodulators, the following materials weresuccessfully on at least one sample:

    ______________________________________                                        NAME            CATEGORY                                                      ______________________________________                                        Lipid A         lipid, bacterial product                                      Ionophore A 23187                                                                             pharmacological stimulant                                     Mixed Bloods    foreign cells, complement, immune                                             complex formation etc.                                        Cycloheximide   DNA, RNA inhibitors, antibiotic                               Snake Venom     immunologically active,                                                       procoagulant                                                  Nicotine        Vasoactive biochemical                                        Salmonella & Other                                                                            bacterial products                                            Endotoxins                                                                    Adenosine Diphosphate                                                                         effects cellular energy balance                                               platelet aggregation                                          Mixed Plasma    foreign species                                               Tuftsin         tetrapeptide - leukocyte                                                      stimulant                                                     Glucan          yeast cell wall, foreign surface                              Streptokinase   clot dissolving agent, lyte                                                   agent, immunologically active                                 Urokinase       clot dissolving agent, lyte                                                   agent, immunologically active                                 Papain          enzyme                                                        Immunoglobulin G (IgG)                                                                        inflammatory blood product                                    Ferritin        foreign protein                                               PPD             purified protein derivative                                   Phytohemagluttinin                                                                            lectin                                                        Pathophysiological Agents                                                                     damaged cells, etc.                                           ______________________________________                                    

EXAMPLE IX

In this Example, the effect of varying immunomodulator concentrations onrecalcification times was measured. The immunomodulators, and otherparameters of testing are set forth below:

A. Thromboplastin, (also called tissue factor of clotting factor III).Material generated by monocytes, procoagulant, immunomodulator.

    ______________________________________                                        Concentration of tissue factor (ug/cc CWB)                                           0       3         6         15                                         ______________________________________                                        RT ± SD                                                                             6.11 ± 0.92                                                                          2.98 ± .60                                                                           2.34 ± .35                                                                         1.96 ± .51                            ______________________________________                                         n = 8 samples                                                            

Significance ANOVA p<:05 for comparisons between different groups.

B. E. coli endotoxin

    ______________________________________                                        Concentration of endotoxin (ug/cc CWB)                                               0       10        1.0       0.1                                               (A)     (B)       (C)       (D)                                        ______________________________________                                        RT ± DF                                                                             5.25 ± 1.03                                                                          3.75 ± .78                                                                           4.20 ± .49                                                                         4.16 ± .32                            ______________________________________                                         n = 8 samples                                                                 Significance (ANUA)                                                           A vs B; P < .001                                                              A vs C; P < 05                                                                A vs D; P < .05                                                               B vs C; P = NS                                                                C vs D; P = NS                                                           

By varying immunomodulator concentrations, the threshold quantitynecessary to give a significant change in RT relative to that of thesaline value may be of clinical significance. The minimum concentrationof a particular immunomodulator necessary to produce change is calledthreshold value and this may vary between diseases and may enable adifferential diagnosis (e.g. cancer, not diabetes, etc.) to be made.

EXAMPLE X USE OF TEST IN MONITORING DRUG THERAPY

Samples of citrated whole blood from patients (4) on Warfarin(anticoagulant) therapy were analyzed with this test. The prothrobintime of patients ranged between 18.6 to 65 seconds (normal 12 sec.).

    ______________________________________                                        RT saline (min)                                                                             RT endotoxin (min)                                              ______________________________________                                        18.7 ± 3.56                                                                              5.48 ± 2.91                                                  ______________________________________                                         significance, paired t test, P < .02                                     

The RT saline is very prolonged due to the anticoagulant drug. However,RT endotoxin mean is in the normal range suggesting that thisanticoagulant has little effect on tissue factor generation.

Advantages of the present invention are many, e.g. donor blood may bepre-screened, particularly where a pathological condition of AIDS mayexist, let alone the undesirability of use of blood for transfusionswhere such blood evidences the existence of a pathological state orcondition in the blood donor. The present invention may be used toevaluate compatibility of transfusion of a particular blood donor. Stillfurther, the methods of the present invention permit the facilemonitoring of the effectiveness of drug therapy or regime to aparticular pathological condition or state in a mammal, e.g. diabetes.The present invention permits a facile evaluation of the potentialacceptance or rejection by a mammal of a transplant organ.

While the present invention is discussed with primary reference to theevaluation of a cellular hematologic fluid to determine if a mammal hasa pre-existing pathological condition, it is apparent to one skilled inthe art that the method of the present invention may be used in theprognosis of treatment of a known pathogenic state in a mammal. Aillustrated, in Example II above, the immune response system of thecellular hematologic fluid of a mammal having undergone surgery for theremoval of cancerous tissue may be evaluated to determine if allcancerous tissue has been removed from the mammal and/or the extent ofsepsis thereof. Similarly, a post-operative protocol, e.g. chemotherapy,may be monitored for effectiveness of such post-operative protocol.

While the invention herein has been described in connection withexemplary embodiments thereof, it will be understood that manymodifications will be apparent to those of ordinary skill in the art andthat this application is intended to cover any adaptations or variationsthereof. Therefore, it is manifestly intended that this invention beonly limited to the claims and the equivalents thereof.

What is claimed is:
 1. A method for analyzing the blood of a mammal todetermine the presence or development of pathology suspected of causingabnormalities in the immune response system and/or the blood coagulationof the mammal consisting essentially of:A. preparing a quantity ofanticoagulated whole blood from a sample of whole blood taken from; B.taking an aliquot portion of said anticoagulated blood and introducingsaid aliquot portion into a first container having therein a vehicle,and thereby preparing a control sample; C. taking a further aliquotportion of said anticoagulated blood and introducing said furtheraliquot portion into a second container having therein immunomodulatorand a vehicle and thereby preparing an activated sample; D. incubatingsaid control sample and said activated sample at a predeterminedsuitable incubation temperature from about 1 to about 4 hours; E.initiating clotting activity and measuring a reaction parameter for eachof said control sample and said activated sample; and F. identifying thepresence of pathology by comparing the reaction parameters measured inStep E with similar reaction parameters measured from a mammal in ahealthy state.
 2. The method of claim 1, wherein said anticoagulatedblood is prepared by mixing a sample of the blood taken from said mammalwith an anticoagulant selected from the group consisting of sodiumcitrate and sodium oxalate.
 3. The method of claim 1, wherein saidimmunomodulator is present in an amount of about 20 ug/cc of a mg/ccsolution or suspension of citrated whole blood.
 4. The method of claim1, wherein said suitable vehicles comprise a quantity of physiologicalsaline solution.
 5. The method of claim 1, wherein said mammal is a homosapien.
 6. The method of claim 1, wherein said immunomodulator isselected from the group consisting of endotoxins, virus, Interferon,phorbol esters, collagens, anticoagulants platelet activating factors,carrageenans, adjunct peptides thromboplastins, antigens, myelin, gramnegative bacteria, lectins and mitogens.
 7. The method of claim 6,wherein said immunomodulator is an endotoxin.
 8. The method of claim 1wherein said predetermined temperature ranges from about 35° C. to about40° C.
 9. The method of claim 8, wherein said predetermined temperatureis approximately 37° C.
 10. A method for monitoring the current statusof a pathological state in a mammal, comprising:K. analyzing the bloodof said mammal wherein said pathological state is present in accordancewith the method of claim 1, to determine the reaction parameters forsaid known pathological state; L. analyzing a further sample of theblood of said mammal at a predetermined time interval also in accordancewith the method of claim 1, to determine the reaction parametersthereof; and M. comparing the reaction parameters of Steps K and L todetermine said current status in said mammal.
 11. The method of claim10, wherein the comparison of Step M is performed by:Q. determining theThrombotic Index (TI) of said mammal's blood derived from the respectivereaction parameters gathered from each of Steps K and L, said ThromboticIndex comprising the ratio of the recalcification time of said controlsample (RT_(v)) to the recalcification time of said activated sample(RTi), (RI=RTv/RTi); and R. comparing the Thrombotic Index derived fromthe reaction parameters of Step K with the Thrombotic Index derived fromthe reaction parameters of Step L.
 12. The method of 10, wherein thecomparison of Step M is performed by:S. determining the PercentageDifference of Clotting (PDOC) of said mammal's blood derived from therespective reaction parameters gathered from each of Steps K and L, saidPDOC comprising the difference between the recalcification times of saidcontrol sample (RTv) and said activated sample (RTi), said differencemultiplied by 100 and divided by the said control sample recalcificationtime. PDOC (RTv-RTi)×100÷RTv; and T. comparing the PDOC value derivedfrom the reaction parameters of Step K with the PDOC value derived fromthe reaction parameters of Step L.
 13. A method for assessing theeffectiveness of corrective surgery on a mammal having an operativepathologic state, which comprises:N. analyzing the blood of said mammalprior to said corrective surgery in accordance with the method of claim1, to determine the reaction parameters thereof; O. analyzing a furthersample of the blood of said mammal taken after said corrective surgery,also in accordance with the method of claim 1, to determine the reactionparameters thereof; and P. comparing the reaction parameters of Steps Aand B to determine the effectiveness of said corrective surgery.
 14. Themethod of claim 13, wherein the comparison of Step P is performed by:Q.determining the Thrombotic Index (TI) of said mammal's blood derivedfrom the respective reaction parameters gathered from each of Steps Nand O, said Thrombotic Index comprising the ratio of the recalcificationtime of said control sample (RTv) to the recalcification time of saidactivated sample (RTi), (TI=RTv/RTi); and R. comparing the ThromboticIndex derived from the reaction parameters of Step N with the ThromboticIndex derived from the reaction parameters of Step O.
 15. The method ofclaim 13, wherein the comparison of Step P is performed by:S.determining the Percentage Difference of Clotting (PDOC) of saidmammal's blood derived from the respective reaction parameters gatheredfrom each of Steps N and O, said PDOC comprising the difference betweenthe recalcification times of said control sample (RTv) and saidactivated sample (RTi), said difference multiplied by 100 and divided bythe said control sample recalcification time, PDOC (RTv-RTI)×100÷RTv;and T. comparing the PDOC value derived from the reaction parameters ofStep N with the PDOC value derived from the reaction parameters of StepO.
 16. The method of claim 1, wherein said reaction parameters areclotting parameters thereof.
 17. The method of claim 16, wherein saidclotting parameters are recalcification times as determined by fibrinformation.
 18. The method of claim 17, wherein clotting activity in StepE is initiated by adding to the samples a compound containing calciumions.
 19. The method of claim 17, wherein the comparison of Step F isperformed by:G. determining the Thrombotic Index (TI) of said mammal'sblood, comprising the ratio of the recalcification time of said controlsample (RT_(v)) to the recalcification time of said activated sample(RT_(i)), (RI=RT_(v) /RT_(i)); and H. comparing the Thrombotic Indexdetermined in Step A with one or more Thrombotic Indices previouslydetermined from ratios derived from the recalcification times ofstandard control samples and corresponding activated samples derivedfrom healthy mammals.
 20. The method of claim 17, wherein the comparisonof Step F is performed by:I. determining the Percentage Difference ofClotting (PDOC) of said mammal's blood, comprising the differencebetween the recalcification times of said control sample (RT_(v)) andsaid activated sample (RT_(i)), said difference multiplied by 100 anddivided by the said control sample recalcification time, PDOC=(RT_(v)-RT_(i))×100÷RT_(v) ; and J. comparing the Percentage Difference ofClotting (PDOC) determined in Step A with standard PDOC valuespreviously determined from the correlation of the recalcification timesof standard control samples and corresponding activated samples derivedfrom healthy mammals.